Purification of thymidine kinase

ABSTRACT

Purified thymidine kinase is recovered from its contaminated aqueous solutions by adsorption on cross-linked dextran of chromatographic grade activated with bromine cyanide and coupled with 3&#39;&#39;- or 5&#39;&#39;-aminothymidine or its N-aminoalkyl derivatives, followed by elution with dilute sodium chloride solution at pH 8.

United States Patent [191 Rohde et al.

[451 Feb. 12, 1974 PURIFICATION OF THYMIDINE KINASE [75] Inventors:Wolfgang Rohde, Bovenden; Axel Lezius, Gottingen, both of Germany [73]Assignee: Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften e.V.,Gottingen, Germany 22 Filed: May 28,1971

21 App]. No.: 148,189

[30] Foreign Application Priority Data June 10, 1970 Germany 2028588[52] US. Cl; 195/66 R, 195/68, l95/DIG. ll [51] Int. Cl C07g 7/02 [58]Field of Search 195/66, 68, 63

[56] References Cited UNITED STATES PATENTS 3,208,918 9/1965 Beers, Jr.195/66 R 3,645,852 2/1972 Axen et al. 195/68 OTHER PUBLICATIONS Okazakiet al., Deoxythymidine Kinase of Escherichia coll, Journal of BiologicalChemistry, Vol. 239, No. 1, 1964 (pp. 269-284) QP501J7.

Primary Examiner-David M. Naff [57] ABSTRACT 6 Claims, No DrawingsPURIFICATION OF THYMIDINE KINASE The present invention is concerned witha new process for obtaining thymidine kinase from aqueous solutions,especially from solutions which are contaminated with other proteins.

Thymidine kinase is an enzyme which is widely distributed in the animaland vegetable kingdoms and can be obtained from the solutions obtainedby the digestion of cellular material. According to a known process,thymidine kinase is obtained from Escherichia coli by digestion at pH 7,a heating step at 70 to 72C., Streptomycin precipitation, two ammoniumsulphate fractionations and chromatography over DEAE- cellulose (R.Okazaki and A. Kornberg in J. Biol. Chem., 239, (1964), p. 269, etseq.). The disadvantage of this process consists in that it iscomparatively laborious and only gives yields of about 7 percent.

A process has now been found which, in an extremely simple manner,enables, in a single step, the enrichment and purification of thymidinekinase with yields of over 90 percent.

The process according to the invention for the purification or recoveryof thymidine kinase consists in that a thymidine kinase-containingaqueous solution is treated with a cross-linked polymer or gel whichcontains bound or 3'-amino-thymidine or N-mono-(wamino-alkyl) derivativethereof, the polymer or gel is separated from the solution and thethymidine kinase eluted.

The invention is based upon the surprisingly found fact that3'-amino-thymdine, and 5-amino-thymidine, as well as their derivativesmonosubstituted on the amino nitrogen by an w-aminoalkyl group, atcomparatively high concentrations of IO M, exert a mild inhibitingaction for thymidine kinase. Further investigation showed that a polymeror gel combined with these compounds and having a porous structure, wasable practically quantitatively and selectively to hold back thymidinekinase from aqueous solutions thereof. A commercially availablecross-linked dextran for chromatographic purposes has hereby proved tobe especially useful.

The preparation of the substituted dextran preferred for the process ofthe invention takes place in a manner known per se for reactions of thistype by activation of the dextran with cyanobromide and subsequentcoupling of the activated dextran with the abovementionedamino-thymidine.

For the carrying out of the process according to the invention, thereare suitable, in principle, all aqueous solutions which containthymidine kinase, with the proviso that no materials are present thereinwhich are capable of reacting with the gel or polymer. The processaccording to the invention is especially suitable for the recovery andpurification of thymidine kinase from the solutions obtained by thedigestion of animal and vegetable, especially of microbiological,starting material. The process according to the invention is alsosuitable for incorporation at any desired point into processes for theobtaining of other proteins since other enzyme activities are hereby notimpaired.

Since Escherichia coli represents an especially suitable startingmaterial for the obtaining of thymidine kinase, there is preferably useda solution obtained by digestion of E. coli. However, other startingmaterials can also be used for obtaining it or synthetically preparedsolutions can be employed.

If solutions obtained by digestion of E. coli are used, it is expedientfirst to carry out a streptomycin precipitation. If value is placed uponthe obtaining of other enzyme activities present in the solution, afurther purification can additionally also take place by a heating stepat about C. Such a heating step has the advantage that the bindingcapacity of the gel or polymer is hereby increased.

. The selective adsorption of the thymidine kinase depends not only uponthe salt concentration of the solution but also upon other processvariables, especially upon the pre-treatment of the substituted dextran.A complete binding of the thymidine kinase is generally achieved whenthe solution has a salt concentration of less than 0.1M. In order toexclude a damaging of the activity of the enzyme, the process is carriedout at a neutral or weakly basic pH value, preferably between pH 7 and8.5. The salt concentration permissible in any particular case can beascertained in an extremely simple manner in that, after the addition ofthe dextran to the solution, the residual activity in the supernatant isdetermined and further water possibly added thereto.

After the adsorption on of the thymidine kinase, the substituted dextranis expediently washed. For this purpose, there are especially suitablesalt solutions with a concentration between about 0.1 and 0.25 M.However, under certain circumstances, substantially higher saltconcentrations can also be used for the washing, for example, in thecase of the use of a dextran column, which has been equilibrated withdilute triethanolamine acetate buffer of pH 7.5. In this case, foreignprotein can still be washed out with 1M salt solutions, for examplethose of sodium acetate, without impairment of the enzyme activity.

The recovery of the bound thymidine kinase (within the scope of theinvention, there is hereby understood not only thymidine kinase but alsodesoxythymidine kinase) takes place by elution of the dextran withhigher salt concentrations, expediently with salt concentrations of atleast 0.3M. A 0.4 to 1.0M, especially 0.5M, NaCl solution of pH 7 to 9,especially of pH 8, has proved to be especially suitable.

The bringing together of the aqueous solution of thymidine kinase withthe substituted dextran can take place in any desired manner.'Expediently, either the dextran is stirred with the solution and thenfiltered off or the solution is filtered over the dextran. In the lattercase, it is preferable to operate in the manner of a columnchromatography.

The process according to the invention enables, with yields of percentand above, an enrichment of the thymidine kinase in a single step up to1,000 fold. Quite apart from these high yields and the extraordinarypurification action, the process is characterised also by its generalapplicability, as well as by the simple preparation of the substituteddextran and the good stability thereof, which permits a repeated usewithout loss of capacity.

The following Examples further illustrate the process of the invention.

A PREPARATION OF THE SUBSTITUTED DEXTRAN Commercially availablecross-linked dextran of chromatographic quality (Sepharose 4B) is pastedwith 3 4 water and then mixed with 100 mg. cyanobromide/ml.desoxythymidine kinase, which was contaminated by dextran, dissolved inthe same volume of water. The pH accompanying proteins, was applied, inthe abovevalue is adjusted to about 11 by the addition of sodiumdescribed manner, to a column of the same volume and hydroxide solutionimmediately and after about 8 mineluted as described in Example 1 TableII shows the utes, the aqueous solution is filtered off with suction 5values of the starting solution of the eluate.

TABLE fi ml. units/ml. 2 U protein mg/ml spec. act. U/mg.

Fraction lll 50000 0.0l08(!) 540 3.13 0.0035 Eluate (concentrated by 805.83 465 6.7 0.87 ultra-filtration) Yield 86% Enrichment 249 fold andthe dextran washed with dilute sodium bicarbonate The above values shewthat the process according to solution. the invention permits theobtaining of thymidine kinase 950 ml. of the pretreated Sepharose aremixed in also from extraordinarily diluted solutions in extremely 0.1Mphosphate buffer of pH 9 with 1.3 g. 5'-aminospecific manner. thymidine.The product thus obtained can be used di- Th above ro e was re eatedwith the use of rectly. v cross-linked dextrans which were modified with3'- amino-thymidine or with N-(e-amino-hexamethylene)- CARRYING OUT OFTHE PROCESS OF THE amino-(5) -thymidine. Comaprable results wereINVENTION hereby obtained.

E. coli are digested in per se known manner in 0.05M W C m?glycyl-glycine buffer of pH 7 by shaking up with glass 1. A process forrecovering purified thymidine kiballs and centrifuging. The centrifugesupernatant is nase, derived from Escherichia coli, from an aqueousmixed with about 0.2 volumes of 5 percent streptomysolution thereofwhich comprises: cin sulphate solution and the precipitate formed iscena. bonding a compound selected from the group contrifuged off. Thesupernatant is heated to 70 to 72C. sisting of y y for 5 minutes andcentrifuged off from insolubles. and N-mono-(w-aminoalkyl) derivativesof said 14.7 litres of a solution obtained in this way, with aamino-thymidines to a porous carrier, said carrier protein content of8.9 mg./ml. and a specific activity of being a cross-linked polymer orgel insoluble in wa- 0.020l U/mg., are subsequently applied to a columnter; which is filled with the substituted dextran described COntactingan aqueous liquid containing said thyunder A. The column is washed withtris buffer of pH midinekinase with said carrier having said com- 8ofionic strength 0.01 and then eluted with 0.02M tris pound bondedthereto until said thymidine kinase buffer of pH 8.0/0.5M NaCl. Table lshews the results. is adsorbed on said carrier; and

" "TABLE I volume ml. units per ml. 2 units protein mg/ml spec. act.U/mg. enrichment yield starting 14700 0.184 2700 8.9 0.0201 solutionclute 1790 1.34 2400 0.32 4.2 204 fold 89% For comparison, with theuseofthe saineEiEEtTnEeB c. eluting the adsorbed thymidine kinase fromsaid lution described in the process according to J. Biol. carrier.

Chem., 239 (1964), p. 269, two ammonium sulphate 5O 2. A process as setforth in claim 1, wherein said carfractionations are carried out andsubsequently a chrorier is dextran activated by means of cyanobromide,matography on DEAE-cellulose. In the following, yield 'and said aqueousliquid has a pH value between 7 and and enrichment according this knownprocess are com- 8.5.

pared with values ofthe process according to the inven- 3. A process asset forth in claim 2, wherein said tion: aqueous solution is purifiedprior to said contacting by precipitation of impurities by means ofstreptomycin sulfate.

4. A process as set forth in claim 2, wherein said aqueous liquidcontains salts in a concentration of not known process (three steps)Yield Enrichment more than 0.1 M. Pmcess accmd'ng 95% 218 Md 5. Aprocess as set forth in claim 2, wherein said adinvention (one step) 89%204 fold -sorbed thymidine kinase 18 eluted from said carrier by meansof a salt solution at least 0.3 molar. 6. A process as set forth inclaim 2, wherein said ad- E mple 2 sorbed thymidine kinase is elutedfrom said carrier by The process according to the invention can also bemeans of an aqueous sodium chloride solution having carried out inextremely dilute solutions. Thus, an a pH value of 7 to 9, and being 0.4to 1.0 normal with aqueous solution with a content of about l/lOO unitsof respect to said sodium chloride.

2. A process as set forth in claim 1, wherein said carrier is dextranactivated by means of cyanobromide, and said aqueous liquid has a pHvalue between 7 and 8.5.
 3. A process as set forth in claim 2, whereinsaid aqueous solution is purified prior to said contacting byprecipitation of impurities by means of streptomycin sulfate.
 4. Aprocess as set forth in claim 2, wherein said aqueous liquid containssalts in a concentration of not more than 0.1 M.
 5. A process as setforth in claim 2, wherein said adsorbed thymidine kinase is eluted fromsaid carrier by means of a salt solution at least 0.3 molar.
 6. Aprocess as set forth in claim 2, wherein said adsorbed thymidine kinaseis eluted from said carrier by means of an aqueous sodium chloridesolution having a pH value of 7 to 9, and being 0.4 to 1.0 normal withrespect to said sodium chloride.